Mechanisms of Single Cell Injury Repair in Migrating Cells
In motile cells, membrane pressure at the membrane/cytoplasm interface is balanced by the cytoskeletal forces and hydrostatic pressure. In particular, it is believed that protrusion of the lamellipodia is controlled by the balance of membrane pressure and the pressure generated by actin polymerization, while bleb formation results from the balance of membrane pressure and hydrostatic pressure. However, exact contributions of these forces to the cell shape and motion are not known. Membrane pressure is a product of tension and local curvature of the membrane; thus, to know the membrane pressure one has to know the exact three-dimensional shape of the cell. We have developed a simple approach to measure cell vertical profile and volume, which is based on the displacement of the fluorescent cell-impermeable dye dissolved in the medium. To get insight into the balance of forces at the membrane interface, we measure membrane tension (with tether assay), protrusion velocity, vertical profile, and volume dynamics in migrating fish epidermal keratocytes subjected to cytoskeletal drugs and volume perturbations. Inhibiting myosin-dependent contraction with blebbistatin resulted in irregular cell shape, and reduced protrusion velocity and membrane tension (variable and low tether forces as compared to a force distribution centered around 35-40 pN in control cells).
Remarkably, hypoosmotic treatment normalized blebbistatin-treated cells by restoring their tension, protrusion velocity, and shape, and also increased tension and protrusion velocity in control cells. Vertical profile measurements showed that hypoosmotic treatment induced persistent cell swelling and substantial increase in the height of the lamellipodia.
We propose that hydrostatic pressure that could be generated due to either myosin dependent contraction or osmotic gradient helps to maintains optimal vertical profile of the lamellipodium and thus cooperates with actin-dependent protrusion.
Keywords: Migrating Cells, Membrane Tension, Hydrostatic Pressure, Actin Polymerizationback